Consumption of cocoa and dark chocolate products has been associated with positive health outcomes including reduced onset of cardiovascular disease, inflammation, diabetes, obesity, and platelet disorders. Cocoa polyphenols, putatively responsible for these beneficial activities, are highly impacted by cocoa variety, agronomic effects and processing history. However, the difference in polyphenol concentration and composition between cocoa products originating from different hybrid clones (selected for high yield) or from different fermentation conditions is not fully understood. Detailed polyphenol characterization including determination of total polyphenol and total procyanidin concentrations, and qualitative and quantitative analysis of (mean) degree of polymerization was conducted. Significant differences in total polyphenol and procyanidin concentrations were observed between five genetic clones grown by the USDA-ARS Cocoa Germplasm Repository located in Mayagüez, Puerto Rico. To facilitate cocoa fermentation research in laboratories distant from cocoa harvesting sites, a laboratory-scale cocoa fermentation model system was developed in this study. This model system used dried, unfermented, cocoa beans and simulated pulp medium as the starting material. The model system supported growth of the essential succession of cocoa fermenting microorganisms and generated similar chemical changes to those observed in on-farm cocoa fermentation. Using this model system, the impact of inoculation with proprietary yeast strains Saccharomyces cerevisiae Lev F and Saccharomyces cerevisiae Lev B on cocoa polyphenol concentration and composition was evaluated. Inoculation with both yeast strains resulted in increased fermentation rate and Lev B inoculation resulted in higher total polyphenol and procyandin contents at the end of fermentation. Overall, the present work addressed the influence of cocoa variety selection and fermentation process conditions on the composition and concentration of polyphenols. These findings will contribute to continued efforts to develop cocoa products with optimized bioactivity and maximum disease preventative effects. / PHD / Annual worldwide cocoa production exceeds four million tons and the cocoa market impacts not only cocoa growing regions, but also countries producing cocoa products. Consumption of cocoa and dark chocolate products has been associated with positive health outcomes including reduced onset of cardiovascular disease, inflammation, diabetes, obesity, and platelet disorders. Cocoa polyphenols, compounds increasingly associated with health benefits, are highly impacted by the variety and processing history of cocoa. However, the extent to which cocoa genetic variety and processing conditions impact polyphenol concentration and composition is not fully understood. Polyphenol characterization was conducted for five genetic varieties sourced from the USDA cocoa germplasm repository located in Mayagüez, Puerto Rico and it was found that genetic variance could contribute to differences in polyphenol concentration. The main focus in this thesis was to develop laboratory cocoa fermentation model system to facilitate cocoa research in laboratories distant from the cocoa growing region. This model system used dried, unfermented cocoa beans and simulated pulp medium as the starting material. The model system was effective in that it supported growth of the essential succession of coca fermenting microorganisms and generated similar chemical changes to those observed in on-farm cocoa fermentation. This model system could be further applied to test the impact of fermentation conditions on cocoa bean quality. The potential for using yeast starter cultures to improve control and consistency of cocoa fermentation was evaluated using the model fermentation system. Yeast inoculation improved fermentation rate, and polyphenol concentration in fermented beans was higher when cocoa fermentation was inoculated with the yeast strain Saccharomyces cerevisiae Lev B. Overall, the present work addressed the influence of cocoa variety selection and fermentation process conditions on the composition and concentration of polyphenols. These findings will contribute to continued efforts to develop cocoa products optimized for maximum health benefits.

La viticulture dépend des conditions climatiques. Dans le contexte de réchauffement climatique, les changements de la vigne et du raisin sous l’effet des températures élevées vraisemblables pour les prochaines décennies pourraient modifier l’aire de répartition des cépages et même menacer la durabilité de la viticulture des régions chaudes. L’objectif de cette étude était donc d’analyser les effets de l’élévation de température sur la composition du raisin, du transcriptome au métabolome. L’utilisation d’un système « open top » passif au vignoble a permis d’augmenter la température autour des grappes de 0.5-1.6 °C en moyenne, une valeur compatible avec le réchauffement climatique prévisible. Les expérimentations ont été conduites sur Cabernet Sauvignon (CS) et Sauvignon Blanc (SB), de la nouaison à la surmaturité, à Bordeaux (France) et en Barossa Valley (Australie). Les analyses ont ciblé essentiellement les métabolites primaires, les composés phénoliques et aromatiques (l’IBMP, arôme de poivron vert; les précurseurs de 3SH, arôme de pamplemousse et la ß-damascenone, arômes floraux). En complément, des analyses RNA-seq et q-PCR ont été réalisées pour explorer la réponse transcriptomique à cet échauffement modéré en conditions réalistes de vignoble.L’échauffement modéré a peu affecté les concentrations en sucres, acides et total en acides aminés, mais a modifié la distribution des différents acides aminés. La composition en acides aminés s’est principalement différenciée suivant la variété, le stade de développement et le site expérimental.Les concentrations finales en IBMP n’ont pas été affectées par l’échauffement. Cependant, à la fermeture de la grappe, les baies de CS échauffées avaient une moindre concentration en IBMP associée à une sous-expression de VviOMT3. La concentration en IBMP des baies de SB échauffées n’a pas montré de différenciation, bien que les niveaux d’expression de VviOMT3 et VviOMT4 soient diminués. Les effets limités et dépendant du génotype suggèrent qu’une augmentation modérée de la température ne serait pas suffisante pour modifier significativement l’IBMP.Glut-3SH-Al était bien plus concentrée que Glut-3SH et Cys-3SH. le traitement échauffé a fait diminuer la présence de Glut-3SH-Al et Cys-3SH dans les baies de SB, en association avec une sous-expression de VviGST4. Par ailleurs, VIT_08s0007g01420 (GSTU8) a été réprimé par le traitement, et pourrait donc être un gène candidat potentiel impliqué dans la biosynthèse de précurseurs de 3SH.Pour les baies de CS, les concentrations en caroténoïdes totaux et celles des deux caroténoïdes majeurs (la lutéine et le β-carotène) n’ont pas réagi à l’augmentation de température. La zéaxanthine a montré une tendance à la diminution sous l’effet de l’échauffement, jusqu’à une diminution significative. Cette concentration plus faible pourrait limiter la biosynthèse de β-damascenone et expliquer la plus faible teneur en β-damascenone observée dans les baies à sur-maturité en cas de température élevée.Un total de 357 gènes (DEGs) ont été différentiellement exprimés en réponse à l’augmentation de température pour les échantillons de 2015 à Bordeaux. D’après l’analyse d’enrichissement « Gene Ontology », le traitement a principalement régulé quatre catégories en relation avec les microtubules, la paroi cellulaire, l’espace extracellulaire et l’activité des facteurs de transcription. 6 DEGs liés à la biosynthèse des anthocyanes ont été régulés négativement, ce qui pourrait expliquer, au moins en partie, la concentration réduite en anthocyanes totaux observée dans les baies de CS échauffées. En revanche, les tanins n’ont pas été affectés par l’augmentation de la température. / Viticulture depends on climate conditions during the growing season. In the context of global warming, any changes in viticulture caused by the rising temperatures expected for the next decades may alter the geographical distribution of grape varieties and even threaten the sustainability of viticulture in hot areas. The objective of this research was to investigate the effects of moderately elevated temperature on grape composition, both at metabolic and transcriptomic levels. A passive open-top heating system was applied in Cabernet Sauvignon (CS) and Sauvignon Blanc (SB) vines grown with standard practice in Bordeaux, France and the Barossa Valley, Australia (CS only) to increase the bunch zone mean temperature by around 0.5-1.6 °C, which was commensurate with the projected global warming. This moderate heating was applied from fruit-set to two weeks after harvest. Metabolites related to technical, phenolic and aromatic maturities (IBMP, the green pepper aroma, precursors of 3SH, grapefruit aroma, and β-damascenone, floral aroma) were assessed, together with transcriptome analysis via RNA-seq and q-PCR, in order to obtain a comprehensive view of berry responses to this moderately elevated temperature in realistic vineyard conditions.The moderately elevated temperature hardly affected the concentrations of sugars, organic acids and total amino acids, but it altered free amino acid composition depending on varieties, vintages and locations.The final concentrations of IBMP were not affected by warming condition in mature berries. However, the elevated temperature significantly reduced IBMP content and expression level of VviOMT3 (a known key gene of IBMP) in CS berries at bunch closure stage, while it reduced the expression levels of VviOMT3 and VviOMT4 at bunch closure stage without affecting IBMP concentration in SB berries. This limited and genotype-dependent effect of elevated temperature suggests that a moderate temperature elevation may not be sufficient to significantly modify IBMP.Glut-3SH-Al was much more concentrated than Glut-3SH and Cys-3SH. Reduced Glut-3SH-Al and Cys-3SH concentrations were associated with a significantly lower expression level of VviGST4 in heated SB berries. Meanwhile, VIT_08s0007g01420 (GSTU8), was down-regulated by elevated temperature and might be a potential candidate gene involved in the biosynthesis of precursors of 3SH.The concentrations of total carotenoids and two predominant carotenoids (lutein and β-carotene) were not altered by elevated temperature in CS berries, but zeaxanthin was reduced by elevated temperature and was significantly less concentrated at harvest. This lower concentration may limit the biosynthesis of β-damascenone and explain the observed lower β-damascenone concentration in post-ripening berries under elevated temperature.A total of 357 genes were differentially expressed (DEGs) in response to the elevated temperature in Bordeaux samples in 2015. Enrichment analysis of Gene Ontology showed that temperature mainly regulated four GO categories, including microtubule, cell wall, extracellular region, and transcription factor activity. 6 DEGs related to anthocyanins synthesis were down-regulated and it could explain, at least in part, the observed lower total anthocyanins in warmed CS. Conversely, tannins were not affected by elevated temperature.The results provide a better understanding of potential global warming effects on metabolite changes during berry development, along with novel molecular insights into the response of grape berry to moderate heating in vineyard conditions.

Due to their relative chemical stability and ubiquity in the environment, chlorinated organic contaminants such as polychlorinated biphenyls (PCBs) pose significant health risks and enduring remediation challenges. Engineered nanoparticles (NPs) provide a novel platform for sensing/remediation of these toxicants, in addition to the growing use of NPs in many industrial and biomedical applications, but there remains concern for their potential long-term health effects. Research highlighted herein also represents a transdisciplinary approach to address human health challenges associated with exposure to PCBs and NPs. The objectives of this dissertation research are two-fold, 1) to develop effective methods for capture/sensing and remediation of environmental toxicants, and 2) to better understand associated risks and to elucidate relevant protective mechanisms, such as lifestyle-related modulators of environmental disease. Prevalent engineered nanoparticles, including aluminum oxide and titanium dioxide, have been studied to better understand effective nanoparticle dispersion methods for in vitro nanotoxicology studies. This work has served both to effectively stabilize these nanoparticles under physiological conditions and to better understand the associated mechanisms of toxicity, which links these metal nanoparticles to endothelial oxidative stress and inflammation through phosphorylation of key cellular signaling molecules and increased DNA binding of pro-inflammatory NFκB. Surface functionalization, though, is being found to limit potential toxicity and has been utilized in subsequent research. A novel polyphenol-functionalized, NP-based system has been developed which combines the biomimetic binding capabilities of nutrient polyphenols with the separation and heating capabilities of superparamagnetic iron oxide NPs for the capture/sensing of organic contaminants in polluted water sources. Magnetic nanocomposite microparticles (MNMs) incorporating the fluorescent polyphenols quercetin and curcumin exhibit high affinity for model organic pollutants followed by rapid magnetic separation, addressing the need for sustainable pollutant remediation. Further work has been performed to both better understand health concerns associated with environmental toxicants such as PCBs and to determine effective methods for modulating their toxicity. This research has shown that PCB remediation through dechlorination is a viable technique for decreasing endothelial inflammation, although complete dechlorination to biphenyl is necessary to effectively eliminate superoxide production, NFκB activation, and induction of inflammatory markers. Additionally, the nutrient polyphenol EGCG, found in green tea, has been shown to serve as a biomedical modulator of in vivo PCB toxicity by up-regulating a battery of antioxidant enzymes transcriptionally controlled by AhR and Nrf2 proteins.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-07-20T19:32:21Z No. of bitstreams: 1 virginiafernandesbraga.pdf: 2075207 bytes, checksum: 720e5dba0516d172aec1843372dcecc2 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-22T15:25:58Z (GMT) No. of bitstreams: 1 virginiafernandesbraga.pdf: 2075207 bytes, checksum: 720e5dba0516d172aec1843372dcecc2 (MD5) / Made available in DSpace on 2016-07-22T15:25:58Z (GMT). No. of bitstreams: 1 virginiafernandesbraga.pdf: 2075207 bytes, checksum: 720e5dba0516d172aec1843372dcecc2 (MD5) Previous issue date: 2011-04-20 / Pitcairnia albiflos Herb. (Bromeliaceae) atualmente se encontra na lista de espécies ameaçadas de extinção. Essa espécie é endêmica dos afloramentos rochosos do município do Rio de Janeiro, RJ, e vem sofrendo com o pisoteio de alpinistas, queimadas, invasão de gramíneas exóticas e extrativismo vegetal. A micropropagação pode ser utilizada como alternativa às condições de risco em que as populações dessa espécie se encontram submetidas, visando à recomposição de populações ameaçadas em ambiente natural, assim como o abastecimento do mercado consumidor. A etapa final da micropropagação é a aclimatização, período em que as plantas ficam mais susceptíveis e sofrem com o estresse oxidativo devido às mudanças nas condições ambientais. No presente trabalho foram avaliadas as atividades enzimáticas antioxidativas da CAT, SOD, POD, PPO e o conteúdo de prolina, além dos teores de pigmentos fotossintéticos em plantas de Pitcairnia albiflos cultivados in vitro, em meios de cultura contendo duas concentrações de sacarose (15 ou 30 g L-1), tampas não vedadas que permitiam trocas gasosas e frascos vedados com tampas e filme plástico de PVC, que impediam a ventilação. Sob essas condições, as plantas foram cultivadas em meios contendo GA3 ou ANA. Após o período de crescimento in vitro, as plantas foram transferidas para condições ex vitro em casa de vegetação. As análises supra-citadas e a determinação dos teores de carboidratos solúveis totais, sacarose, amido, açúcares redutores, conteúdo relativo de água e suculência também foram realizadas nas plantas previamente cultivadas in vitro com 15 ou 30 g L-1 de sacarose e GA3 em tubos com tampas vedadas, após submissão das mesmas a estresse hídrico durante 24, 38 ou 52 dias. Após o período de estresse hídrico, as plantas foram reidratadas durante 34 dias sob irrigação periódica em casa de vegetação. Nos tecidos cultivados in vitro percebeu-se o surgimento de características de hiperidricidade nas plantas cultivadas com 15 g L-1 de sacarose, GA3 e tubos com tampas vedadas, o que foi evidenciado pelo menor acúmulo de prolina, aumento das atividades das enzimas antioxidantes e menor acúmulo de pigmentos fotossintetizantes. Na condição ex vitro, as plantas cultivadas anteriormente em meio de cultura contendo 15 g L-1 de sacarose apresentaram maior atividade das enzimas antioxidantes não havendo, em alguns casos, diferenças significativas em comparação com a concentração mais elevada de sacarose. Nessa condição o acúmulo de prolina foi menor, o que é indicativo de maior estresse oxidativo nessas plantas durante a aclimatização. Durante o estresse hídrico houve queda na atividade de todas as enzimas estudadas, embora essa queda tenha sido mais acentuada para as plantas que inicialmente foram cultivadas com 15 g L-1 de vi sacarose. O acúmulo de prolina aumentou com o prolongamento do estresse hídrico, sendo maior nas plantas que foram cultivadas in vitro com 30 g L-1 de sacarose. Não houve diferenças significativas no conteúdo de pigmentos fotossintetizantes e nas suas relações para nenhuma das concentrações de sacarose, exceto para os carotenóides totais, que apresentaram aumento significativo ao longo do período de estresse hídrico para as plantas previamente cultivadas com a menor concentração de sacarose. Os conteúdos de carboidratos solúveis totais e sacarose aumentaram com o prolongamento do estresse, sendo mais acentuados na concentração de 30 g L-1 de sacarose. Os conteúdos de amido, o conteúdo relativo de água e a suculência apresentaram redução com o aumento do estresse hídrico. Após a reidratação, todas as plantas mostraram capacidade de recuperação, apresentando valores próximos aos dos controles para todas as variáveis analisadas. Ressalta-se, todavia, que as plantas tratadas com 30 g L-1 de sacarose tiveram melhor recuperação quando comparadas com aquelas que foram tratadas com 15 g L-1 de sacarose. Em função dos resultados obtidos, é possível concluir que a concentração de sacarose utilizada in vitro apresenta influência no processo de aclimatização ex vitro e também, posteriormente, no campo, na capacidade de recuperação das plantas à seca quando elas são submetidas a estresse hídrico. As plantas cultivadas in vitro com 15 g L-1 de sacarose se mostraram mais sensíveis à seca e, possivelmente, não sobreviveriam caso fossem transferidas dos tubos de ensaio diretamente para o campo. As plantas cultivadas in vitro com 30 g L-1 de sacarose aparentemente eram mais resistentes ao processo de aclimatização ex vitro, apresentando maiores chances de sobrevivência em campo, maior tolerância à seca e maior capacidade de recuperação após períodos prolongados de estresse hídrico. / Pitcairnia albiflos Herb. (Bromeliaceae) is currently in the list of endangered species. This species is endemic of the inselbergs of the city of Rio de Janeiro, RJ, and has been suffering with the mountaineer’s trampling, wildfires, invasion of exotic grasses and plant extraction. The micropropagation can be used as an alternative to the risk conditions under which populations of this species are submitted, aiming at the recomposition of endangered populations in the natural environment, as well as the supply of the consumer market. The final stage of the micropropagation is the acclimatization, period in which the plants become more susceptible and suffer from oxidative stress due to the changes in the environmental conditions. In the present study it were evaluated the antioxidative enzymatic activities of CAT, SOD, POD, PPO and proline content, besides the levels of photosynthetic pigments in plants of Pitcairnia albiflos grown in vitro in culture mediums containing two sucrose concentrations (15 or 30 g L-1). Part was covered with unsealed lids that allowed gas exchanges and part was kept in sealed flasks with lids and PVC plastic film, that didn’t allow the ventilation. Under these conditions, the plants were cultivated in culture mediums containing GA3 or NAA. After the in vitro growth period, the plants were transferred to ex vitro conditions at a greenhouse. The above mentioned analyses and the determination of total soluble carbohydrate levels, sucrose, starch, reducer sugars, relative water content and succulence were also performed on the plants previously grown in vitro with 15 or 30 g L-1 of sucrose and GA3 in tubes with sealed lids, after the submission of these to water stress during 24, 38 or 52 days. After the water stress period, the plants were rehydrated for 34 days under regular irrigation at the greenhouse. In the in vitro cultivated tissues it was noted the emergence of hyperhydricity characteristics in the plants grown with 15 g L-1 of sucrose, GA3 and tubes with sealed lids, which was evidenced by the lowest proline accumulation, the increased in the antioxidative enzymatic activities and the lowest accumulation of photosynthetic pigments. In the ex vitro condition, the plants previously grown in culture mediums containing 15 g L-1 of sucrose presented larger antioxidative enzymatic activities, which did not show, in some cases, significant differences compared with the largest concentration of sucrose. In this condition, the proline accumulation was lower, which is an indicative of larger oxidative stress in these plants during acclimatization. During the water stress, there was a fall in the activity of all studied enzymes, although that fall had been more evident in the plants that were initially cultivated with 15 g L-1 of sucrose. The proline accumulation increased with the extension of the water stress, being larger in the plants grown in vitro with 30 g L-1 of sucrose. There were no viii significant differences in the content of photosynthetic pigments and in their relation with any sucrose concentrations, except for total carotenoids, which significantly increased over the period of water stress for the plants previously grown with the lowest concentration of sucrose. The contents of total soluble carbohydrates and sucrose increased with the extension of the stress, being more accentuated in the 30 g L-1 of sucrose concentration. The contents of starch, the relative content of water and succulence presented a reduction with the increase of water stress. After the rehydration, all plants showed recovery capacity, presenting values close to those from the control groups for all the analyzed variables. It should be noted, however, that the plants treated with 30 g L-1 of sucrose had better recovery compared with those that were treated with 15 g L-1 of sucrose. According to the obtained results, it is possible to conclude that the concentration of sucrose used in vitro presents influence on the process of ex vitro acclimatization and also, later in the field, in the recovery capacity of the plants to drought when they are submitted to water stress. The plants grown in vitro with 15 g L-1 of sucrose were more sensitive to drought and, possibly, would not survive if they were transferred from the test tubes directly to the field. The plants grown in vitro with 30 g L-1 of sucrose were apparently more resistant to the ex vitro acclimatization process, presenting greater survival chances in the field, larger drought tolerance and larger recovery capacity after extended periods of water stress.

Le principal objectif de ce travail de thèse était d’étudier l’influence de la fonctionnalisation des protéines par des sucres ou des polyphénols de raisin dans la formulation et le comportement de microsphères de vitamine A. La formulation de différents conjugués issus soit de la réaction de Maillard soit de la complexation des polyphénols sur les protéines a été effectué sur trois matières premières protéiques : les protéines de pois, le caséinate de sodium de lait de vache et la gélatine de type A porcine. Dans une première partie, les caractéristiques et le pouvoir émulsifiant des conjugués ont été étudiés, et ont confirmé le potentiel de stabilisation d’une huile dans le temps. Une seconde partie s’est concentrée sur les observations au microscope électronique à balayage des microsphères et sur une méthodologie d’observation spécifique à ce genre d’échantillon. Une troisième partie a étudié l’influence des fonctionnalisations sur la stabilité de la vitamine A dans le temps, sur sa libération dans des milieux de digestion gastriques et entériques simulés, et sur la libération de géraniol co-encapsulé. La dernière étude a porté sur le potentiel muco-adhésif des microsphères en utilisant une technique d’analyse originale. / The main objective of this thesis was to study the influence of functionalization of proteins by sugars or grape polyphenols in the vitamin A microsphere formulation and behavior. The formulation of different conjugated stemming either from the Maillard reaction or from the complexation of polyphenols on proteins was made on three proteins : pea proteins, sodium caseinate and type A gelatin. In a first part, the characteristics and the emulsifying power of the combined were studied, and confirmed the potential of stabilization of oil in the time. A second part was on the observations with scanning electron microscope of microspheres and on the methodology of specific observation in this kind of sample. The third part studied the influence of functionalization on vitamin A stability in the time, liberation on gastric or enteric digestion media, and liberation of co-encapsulated geraniol. The last study concerned the muco-adhesive potential of microspheres by using an original analysis.

La composition phénolique du vin rouge détermine fortement sa qualité: couleur, goût, texture et la plupart des bienfaits pour la santé. Les conditions ambiantes de la vigne modulent l'équilibre hormonal endogène et l'expression de gènes qui contrôlent la voie de synthèse des composés flavonoïdes, en déterminant la composition phénolique finale du raisin. Même s'ils ont été étudiés, les effets des applications des régulateurs de croissance végétale sur l'équilibre hormonal endogène et la qualité du raisin, les effets de ces substances sur la composition et la qualité du vin sont pauvrement documentés. Le traitement des raisins destinés à la vinification avec des régulateurs de croissance végétale est un outil potentiel pour modifier la qualité des raisins et du vin rouge. Ce projet de thèse a pour objectif d’étudier l’impact d'applications de régulateur de croissance végétale sur la composition phénolique des raisins de Vitis vinifera L. cépages Cabernet Sauvignon et Carménère. L’acide abscissique, l’acide indole-3-acétique et l'acide 2-chloroethilphosphonique ont été appliqués à divers stades phénologiques du raisin, doses et conditions environnementales: Les essais ont été menés à Maipo et Cachapoal au Chili et à Bordeaux en France, dans des vignobles commerciaux et expérimentaux ainsi que sur des plantes cultivées en pots. Il a été examiné l'effet de ces traitements sur le contenu interne d'hormones, sur l'expression de gènes structuraux et régulateurs de la synthèse de composés flavonoides et sur la qualité des raisins, en particulier la composition phénolique de sa pellicule. De plus, des vinifications ont été réalisées à partir de raisins traités pour déterminer l'effet des traitements sur la composition chimique et phénolique du vin, ainsi que sur des attributs qualitatifs tels que les arômes et la texture des vins, jugés par un panel d'évaluation sensorielle. / Phenolic composition strongly determines red wine quality: color, taste, texture and most health benefits. Vineyard environmental conditions modulate endogenous hormonal balance and gene expression which control the flavonoid biosynthetic pathway leading to final grape phenolic composition. Even when the effects of plant growth regulator applications on grape endogenous hormonal balance and quality have been studied, the effect of these substances on wine composition and quality is poorly documented. The treatment of wine grapes with plant growth regulators is a potential tool in order to modify red wine phenolic composition and quality. This thesis project describes six experiments on plant growth regulator applications on developing grapes of Vitis vinifera L., cvs Cabernet Sauvignon and Carménère. Abscisic acid, Indole-3-acetic acid and 2-chloroethylphosphonic acid were applied in different phenological stages, doses and environmental conditions: Maipo and Cachapoal regions in Chile and Bordeaux region in France, commercial and experimental vineyards and plants in containers. The effect on changes in the internal hormonal content, expression of flavonoid biosynthetic and regulatory genes and grape quality, in particular grape skin phenolic composition were examined. In addition, winemaking was performed in order to assess the effect of treatments on wine chemical and phenolic composition and on wine aroma and texture attributes judged by a sensory panel.

Les propriétés antioxydantes du resvératrol sont d’un grand intérêt pour contrer la dysfonction endothéliale où la contribution du stress oxydant est majeure. Cette dysfonction endothéliale est d’ailleurs bien caractérisée en hypertrophie ventriculaire gauche (HVG). Cette étude vise à explorer les effets thérapeutiques du resvératrol sur la dysfonction endothéliale des artères coronaires épicardiques associée à l’HVG. L’HVG est induite suite à un cerclage aortique (CA) sur des porcelets de 2 mois. Le resvératrol est ensuite administré à 20 mg/kg/jour per os aux animaux répertoriés dans différents groupes. Le groupe 1 comprend les animaux contrôles sans CA alors que le groupe 2 représente les porcelets ayant subit le CA sans traitement. Le groupe 3 comprend les animaux traités pendant les 60 jours après la chirurgie alors que le groupe 4 est traité seulement à partir du 30ème jour jusqu’au jour 60. Des analyses échocardiographiques et histologiques ont été effectuées afin de déterminer le degré d’hypertrophie et l’impact du resvératrol sur la progression de l’HVG alors que la réactivité vasculaire a été évaluée par des expériences de chambres d’organes. La dysfonction endothéliale a été étudiée in vitro par quantification des métabolites nitriques et des niveaux de GMPc tandis que le stress oxydant est décrit par les niveaux d’angiotensine II (Ang II) et de protéines carbonylées. Les courbes dose-réponse à la sérotonine du groupe 3 traité pendant les 60 jours au resvératrol ont démontré une relaxation vasculaire significativement améliorée comparé au groupe 2 non-traité (p<0,05). Le pourcentage de changement du rapport de la masse du ventricule gauche sur la masse corporelle (LVmass/BW) a démontré une inhibition du développement de l’HVG dans le groupe 3 alors que le groupe 4 n’a pas eu d’effets bénéfiques des 30 jours de traitement au resvératrol. La biodisponibilité du NO représentée par la mesure de ses métabolites en circulation (398,71±21,74 ; 151,04±14,95 ; 241,86±15,53 ; 208,17±26,52 uM pour les groupes 1, 2, 3 et 4 respectivement, p<0,001 pour groupe 1 et 3 vs groupe 2) et par le niveau de GMPc a été augmentée avec l’administration du polyphénol (2,54±0,63 ; 0,67±0,45 ; 1,71±0,25 ; 1,98±0,29 pmol/ml pour les groupes 1, 2, 3 et 4 respectivement, p<0,05 pour groupe 1, 3 et 4 vs groupe 2). Le rôle antioxydant du resvératrol a été confirmé avec une réduction des niveaux de protéines carbonylées chez le groupe 3 comparé aux valeurs du groupe 2 (0,14±0,05 vs 0,33±0,03 nmol/mg respectivement) sans diminution des niveaux d’Ang II. Le resvératrol a aussi réduit l’hypertrophie des cardiomyocytes et la fibrose interstitielle. Ainsi, le resvératrol peut effectivement réduire la dysfonction endothéliale des artères coronaires épicardiques et limiter l’occurrence de remodelage myocardique associé à l’HVG, principalement à travers l’activation de la signalisation dépendante du NO. / Antioxidant properties of resveratrol are of great interest regarding endothelial dysfunction in which oxidative stress has a major causative role. This dysfunction is well characterized in left ventricular hypertrophy (LVH). The current study was designed to explore the effects of resveratrol as a treatment for the coronary endothelial dysfunction associated with LVH. An aortic banding (AB) was performed on swine to induce LVH on two-month-old swine for 60 days. Resveratrol was administered at 20 mg/kg/d per os to different groups. Group 1 represented healthy controls while group 2 was not treated. After AB, group 3 received resveratrol from day 1 to 60 post-surgery whereas group 4 was treated from day 30 to 60 only. Echocardiographic and histological studies were performed to evaluate the degree of LVH. Vascular reactivity of epicardial coronary arteries was assessed in organ chamber experiments. Endothelial dysfunction was evaluated in vitro by nitric oxide (NO) metabolites and cyclic guanosine-3,5-monophosphate (cGMP) while angiotensin II (Ang II) levels and protein carbonylation evaluated Oxidative stress. After treatment, the LVmass/body weight ratio was improved in group 3 associated with reduced cardiomyocyte hypertrophy and interstitial fibrosis (p<0.05). Dose-response curves to serotonin for the resveratrol treated group 3 demonstrated a greater vascular relaxation compared to LVH untreated group 2 (p<0.05). Bioavailability of NO measured by its circulating metabolites (398,71±21,74; 151,04±14,95 ; 241,86±15,53 ; 208,17±26,52 uM for groups 1, 2, 3 and 4 respectively, p<0,001 for groups 1 and 3 vs group 2) and by vascular cGMP (2,54±0,63 ; 0,67±0,45 ; 1,71±0,25 ; 1,98±0,29 pmol/ml for groups 1, 2, 3 and 4 respectively, p<0,05 for group 1,3 and 4 vs group 2) was increased. The antioxidant effect of the polyphenol was confirmed by decreased protein carbonylation with the 60 days treatment (0,14±0,05 vs 0,33±0,03 nmol/mg for group 3 vs 2) associated to unchanged Ang II levels. Therefore, resveratrol effectively reduces epicardial coronary endothelial dysfunction and limits the occurrence of cardiac remodelling associated with LVH, mainly through an NO-dependent pathway.

Tesis por compendio / [ES] El oscurecimiento de los tejidos de frutas y verduras dañados puede provocar cambios indeseables y el rechazo por parte del consumidor. Este deterioro, conocido como pardeamiento enzimático, es causado principalmente por la enzima polifenol oxidasa (PPO), que oxida los compuestos fenólicos en pigmentos rojizos llamados melanoides. De esta forma, la prevención de la actividad de PPO en el procesamiento postcosecha tanto en frutas y verduras como en sus licuados, ha recibido desde siempre mucha atención por parte de la industria alimentaria. Sin embargo, los tratamientos actuales presentan diversos inconvenientes, entre los que podemos destacar los efectos negativos en la calidad nutricional de los productos y su elevado coste. El cometido de esta tesis doctoral se centra en el desarrollo y evaluación de nuevas estrategias no térmicas para la inhibición de la PPO con el objetivo final de detener el pardeamiento enzimático. Para esto se abordaron dos estrategias diferentes. Por un lado, se seleccionaron y evaluaron varias poliaminas macrocíclicas. Se determinó que la estructura química influye fuertemente en el poder inhibidor, existiendo dos compuestos altamente eficaces contra el pardeamiento enzimático, los cuales presentan IC50 de 10 µM y 0.30 mM. Su eficacia se validó en zumo de manzana recién licuado retrasando el pardeamiento enzimático y la pérdida de compuestos fenólicos totales. Por otro lado, se estudió el desarrollo y aplicación de partículas de sílice mesoporosas funcionalizadas con diversos grupos químicos. Los resultados mostraron que tanto la estructura del material como la funcionalización son determinantes. El soporte UVM-7 ofreció la inhibición más fuerte sobre la enzima PPO, y una vez funcionalizado con grupos tiol este aumentó notablemente su poder inhibidor, deteniendo el pardeamiento enzimático en zumo de manzana. Por el contrario, los grupos amino, aunque mostraron menor poder inhibidor, fueron capaces de inmovilizar a la enzima y eliminarla del medio. Finalmente, el soporte UVM-7 fue magnetizado para su fácil eliminación del medio, evitando así la etapa de filtración. El soporte UVM-7 magnetizado y funcionalizado con tioles logró mantener la concentración inicial de vitamina C y flavonoides en el zumo de manzana. Además, la capacidad antioxidante y el contenido de fenoles totales se mantuvieron casi sin cambios. / [CA] L'enfosquiment de fruïtes i verdures provoca un alt rebuig per part del consumidor. Aquest fet genera grans pèrdues econòmiques i un gran desaprofitament d'aliments. Aquest deteriorament, conegut com enfosquiment enzimàtic, és causat principalment per l'enzim polifenol oxidasa (PPO), que oxida els compostos fenòlics en pigments vermellosos anomenats melanoïdines. D'aquesta manera, la prevenció de l'activitat de la PPO en el processament postcollita tant en fruites i verdures com en els seus liquats, ha rebut des de sempre molta atenció per part de la indústria alimentària. No obstant això, els tractaments actuals presenten diversos inconvenients, entre els quals podem destacar els efectes negatius en la qualitat nutricional dels productes o el seu elevat cost. L'objectiu d'aquesta tesi doctoral es centra en el desenvolupament i avaluació de noves estratègies no tèrmiques per a la inhibició de la PPO amb la finalitat de detindre el enfosquiment enzimàtic. Per això s'aborden dues estratègies diferents. D'una banda, es va seleccionar i avaluar diverses poliamines macrocícliques. Determinat que l'estructura química influeix fortament en el poder inhibidor, existint dos compostos altament eficaços contra el enfosquiment enzimàtic, els quals presenten IC50 de 10 µM i 0.30 mM. La seua eficàcia es va validar en el suc de poma recentment liquat retardant el enfosquiment enzimàtic i la pèrdua de compostos fenòlics totals. D'altra banda, es va estudiat el desenvolupament i aplicació de partícules de sílice mesoporoses funcionalitzades amb diversos grups químics. Els resultats van mostrar que tant l'estructura del material com la funcionalització foren determinants. El suport UVM-7 va oferir la inhibició més forta sobre l'enzim PPO, i una vegada funcionalitzat amb grups tiol aquest va augmentar notablement el seu poder inhibidor, detenint el enfosquiment enzimàtic en el suc de poma. Tot i això, els grups amina, encara que mostren menys poder inhibidor, foren capaços d'immobilitzar l'enzim i eliminar-la del medi. Finalment, el suport UVM-7 va ser magnetitzat per la seva fàcil eliminació del mig, evitant així l'etapa de filtració. El suport UVM-7 magnetitzat i funcionalitzat amb grups tiol va aconseguir mantindre la concentració inicial de vitamina C i flavonoides en el suc de poma. A més, la capacitat antioxidant i el contingut de fenols totals es van mantindre quasi sense canvis. / [EN] The browning of injured fruit and vegetable tissues can cause undesirable changes and consumers' rejection resulting in high economic losses and food waste. This damage, known as enzymatic browning, is mainly caused by the polyphenol oxidase enzyme (PPO) which oxidize the phenolic compounds found in these tissues into reddish pigments named melanonids. Thus, preventing PPO activity in post-harvest processing fruits and vegetables including their juices has received a lot of attention from the food industry. Nonetheless, the current alternatives have some drawbacks such as negative effects on the nutritional quality of products or high cost. Hence, the purpose of this doctoral thesis is focused on the development and evaluation of new non-thermal strategies for PPO inhibition and prevention of the enzymatic browning. For this purpose, two different strategies were addressed; On one hand, several macrocyclic polyamine compounds were selected and evaluated. The chemical structure was found to strongly influence the inhibitor power, and two different compounds were found to be efficient against the enzymatic browning with IC50 of 10 µM and 0.30 mM. Their effectiveness was proved in cloudy apple juice resulting in a delay of the enzymatic browning and the loss of total phenolic compounds. On the other hand, the development and application of mesoporous silica particles functionalized with diverse chemical groups were studied. The results showed that both the structure of the material and the type of functionalization are decisive. The UVM-7 support offered the strongest inhibition of the PPO. The functionalisation with thiol groups enhanced the inhibitor power stopping the enzymatic browning in cloudy apple juice. Alternatively, amine groups, although showing less inhibitory power, were able to immobilise the enzyme. Finally, the UVM-7 support was magnetized for easily elimination of the medium, thus preventing the juice filtration need. Cloudy apple juice treated with magnetized UVM-7 functionalized with tiol groups maintained the initial concentration of both vitamin C and flavonoids. Moreover, the antioxidant capacity and the total phenolic content remained almost unchanged. / Financial support by the Spanish Ministerio de Ciencia, Innovación y Universidades (project RTI2018-100910-B-C44 and MAT2015-64139-C4-1), Ministerio de Economía y Competitividad (projects CTQ2016-78499-C6-1-R, Unidad de Excelencia MDM 2015- 0038 and CTQ2017-90852-REDC) and Generalitat Valenciana (projects PROMETEOII2015-002 PROMETEO/2018/024 and GVA/2014/13) is gratefully acknowledged. / Muñoz Pina, S. (2021). Mesoporous silica particles and macrocyclic ligands as modulators of polyphenol oxidase activity in food systems [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/174934 / Compendio

Tesis por compendio / [ES] El objetivo de esta tesis doctoral ha sido la valorización de los residuos agroindustriales de los frutos de la sandía (Citrullus lanatus) y del caqui (Diospyros kaki Thunb.) para obtener compuestos de alto valor añadido con potenciales aplicaciones tecnológicas en la industria alimentaria. En concreto, la corteza de la sandía y los descartes del caqui se contemplan como una prometedora fuente de pectina, no sólo por la importancia económica de los cultivos, sino también por las interesantes características estructurales de la pectina extraída y sus propiedades funcionales. Además, se sabe que los frutos del caqui son una fuente de compuestos bioactivos que pueden utilizarse para diseñar productos alimentarios más saludables. En la primera parte de esta tesis, se llevó a cabo una metodología de superficie de respuesta para evaluar cómo afectan los diferentes parámetros del proceso a la composición, las características estructurales y el rendimiento de extracción de la pectina de la corteza de la sandía (WRP). A partir de estos resultados, se utilizó una combinación de herramientas reológicas, microestructurales y de dispersión para dilucidar el mecanismo de estabilización de la emulsión de WRP, que fue capaz de retener altas cantidades de aceite de girasol (>60%), evitando la coalescencia y la floculación. A continuación, se evaluó el efecto de la des-ramificación contralada enzimáticamente, la des-esterificación y la eliminación de proteínas, en la composición y la estructura molecular de la WRP, y se analizó en profundidad el potencial de la pectina tratada enzimáticamente para preparar hidrogeles con la adición de CaCl2, con el fin de comprender cómo la estructura de la pectina y la composición del extracto afectaban a la formación y las propiedades del gel. Se observó que las pequeñas cadenas laterales de arabinogalactano presentes en la pectina con bajo grado de esterificación, actuaban como refuerzo, induciendo la formación de redes más densamente empaquetadas y de hidrogeles más fuertes que sus homólogos menos ramificados. La pectina de sandía tratada enzimáticamente y con mejores propiedades de gelificación y rendimiento, se utilizó posteriormente para desarrollar estructuras de aerogeles utilizando secado con CO2 supercrítico. La formación de una estructura compactada durante la formación del aerogel lo convirtió en un buen candidato como sistemas de liberación de compuestos activos. En la segunda parte de la tesis, se estudió la potencial valorización de residuos de biomasa de caqui mediante la extracción de compuestos bioactivos y extractos ricos en pectina y la evaluación de sus propiedades funcionales. Un extracto rico en polifenoles obtenido a escala semi-industrial mostró actividad antiviral y fue capaz de reducir el envejecimiento celular y el contenido de grasa sin afectar al desarrollo del organismo modelo C. elegans. Además, se optimizaron y caracterizaron las condiciones de extracción de ingredientes funcionales a base de pectina y polifenoles, cuya composición dependió de la severidad de las condiciones de extracción. Estos extractos funcionales de pectina mostraron actividad antiviral, probablemente potenciada por la mayor estabilidad de los polifenoles que forman complejos con la pectina y se exploró, de manera exitosa, su aplicación como recubrimiento comestible en arándanos. / [CAT] L'objectiu d'aquesta tesi doctoral va ser la valorització de residus agroindustrials procedents de fruites de síndria (Citrullus lanatus) i caqui (Diospyros kaki Thunb.) per obtenir compostos d'alt valor afegit amb potencials aplicacions tecnològiques en la indústria alimentària. En concret, l'escorça de síndria i els descarts de caqui es preveuen com una prometedora font de pectina, no només per la importància econòmica dels cultius, sinó també per les interessants característiques estructurals de la pectina extreta i les propietats funcionals relacionades. A més, se sap que les fruites de caqui són una font de compostos bioactius que es poden utilitzar per dissenyar productes alimentaris més saludables. En la primera part d'aquesta tesi es va dur a terme una metodologia de superfície de resposta per avaluar com els diferents paràmetres de procés afecten la composició, les característiques estructurals i el rendiment d'extracció de la pectina de síndria (WRP). A partir dels resultats, es va utilitzar una combinació d'eines reològiques, microestructurals i de dispersió per dilucidar el mecanisme estabilitzador de l'emulsió de WRP, que va ser capaç de retenir altes quantitats d'oli de gira-sol (>60%), evitant la coalescència i la floculació. A continuació, es va avaluar l'efecte del de-brassament enzimàtic controlat, la des-esterificació i l'eliminació de proteïnes sobre la composició i l'estructura molecular del WRP i es va analitzar profundament el potencial de la pectina tractada enzimàticament per preparar hidrogels amb l'addició de CaCl2 per entendre com l'estructura de la pectina i la composició de l'extracte afectaven la formació i les propietats del gel. Es va observar que les petites cadenes laterals d¿arabinogalàctans presents a la pectina amb baix grau d'esterificació, actuaven com a reforç, induint la formació de xarxes més densament empaquetades i hidrogels més forts que els seus homòlegs menys ramificats. Posteriorment es va utilitzar la pectina de l'escorça de síndria tractada enzimàticament amb millors propietats gelificants i rendiment per desenvolupar estructures d'aerogel utilitzant CO2 supercrític. La formació d'una estructura compactada durant la formació d'aerogels la va convertir en un bon candidat per als sistemes de alliberació de compostos actius. En la segona part de la tesi es va estudiar la potencial valorització dels residus de biomassa de caqui mitjançant l'extracció de compostos bioactius i extractes rics en pectina i l'avaluació de les seves propietats funcionals. Un extracte obtingut a escala semi-industrialment ric en polifenols va mostrar una activitat antiviral i va ser capaç de reduir l'envelliment cel·lular i el contingut en greixos sense afectar el desenvolupament de l'organisme model de C. elegans. A més, es van optimitzar i caracteritzar les condicions d'extracció d'ingredients funcionals a base de pectina-polifenol, la composició de la qual depenia de la gravetat de les condicions d'extracció. Aquests extractes funcionals de pectina van mostrar una alta eficiència antiviral, probablement millorada per la major estabilitat dels polifenols com a complexos de pectina. De manera exitosa, es va explorar una potencial aplicació com a recobriment comestible sobre nabius. / [EN] The aim of this doctoral thesis was the valorisation of agro-industrial wastes from watermelon (Citrullus lanatus) and persimmon (Diospyros kaki Thunb.) fruits to obtain high added-value compounds with potential technological applications in the food industry. Specifically, watermelon rind and persimmon discards are envisaged as a promising pectin source, not only because of the economic importance of the crops but also because of the interesting structural features of the extracted pectin and related functional properties. Furthermore, persimmon fruits are known to be a source of bioactive compounds which can be used to design healthier food products. In the first part of this thesis, a response surface methodology was carried out to evaluate how different process parameters affect the composition, structural features and extraction yield of watermelon rind pectin (WRP). Based on the results, a combination of rheological, microstructural and scattering tools was used to elucidate the emulsion stabilizing mechanism of WRP, which was able to retain high amounts of sunflower oil (>60%), avoiding coalescence and flocculation. Then, the effect of controlled enzymatic debranching, de-esterification and protein removal on WRP composition and molecular structure was evaluated and the potential of the enzymatically-treated pectin for preparing hydrogels with the addition of CaCl2 was deeply analysed in order to understand how pectin structure and extract composition affected gel formation and properties. It was observed that the small arabinogalactan side chains present in the pectin with low degree of esterification, acted as a reinforcement, inducing the formation of more densely packed networks and stronger hydrogels than their less-branched counterparts. An enzymatically-treated WRP with better gelling properties and yield was subsequently used to develop aerogel structures using supercritical CO2. The formation of a compacted structure during aerogel formation made it a good candidate for drug delivery systems. In the second part of the thesis, the potential valorisation of persimmon biomass waste through the extraction of bioactive compounds and pectin-rich extracts and the evaluation of their functional properties was studied. A semi-industrially scaled-up polyphenol-rich extract showed an effective antiviral efficiency and it was able to reduce the cellular ageing and fat content without affecting the development of C. elegans model organism. Furthermore, the extraction conditions of functional pectin-polyphenol-based ingredients were optimized and characterized, which composition was dependent on the severity of the extraction conditions. These functional pectin extracts showed high antiviral efficiency, probably enhanced by the higher stability of the polyphenols as pectin complexes. A successful potential application was explored with an edible coating on blueberries. / This research was funded by grant RTI-2018-094268-B-C22 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe". Mendez D. A. is supported by the Ministry of Science, Technology and Innovation (Minciencias) of the Colombian Government (783-2017). M. J. Fabra and A. Martinez- Abad are recipients of Ramon y Cajal (RYC-2014-158) and Juan de la Cierva (IJDC-2017- 31255), respectively, from the Spanish Ministry of Economy, Industry and Competitiveness. This work was also funded by the grant INNVAL10-19-009-CA8250 from Agència Valenciana d ́Innovació (AVI). This research is part of the CSIC program for the Spanish Recovery, Transformation and Resilience Plan funded by the Recovery and Resilience Facility of the European Union, established by the Regulation (EU) 2020/2094. Interdisciplinary Platform for Sustainable Plastics towards a Circular Economy+. (PTI-SusPlast+) is also acknowledged for financial support. Project INNEST/2021/27 was granted by Agencia Valenciana de Innovación (AVI) and co- financed by the European Union through the Operative Programme of FEDER from Comunitat Valenciana 2014-2020. We also appreciate the funding of the CERCA program (Centres de Recerca de Catalunya) of the Generalitat de Catalunya and from the CIEN project “BIOPRO” from CDTI. Nune Sahakyan is kindly acknowledged for experimental support / Méndez Reyes, DA. (2022). Valorisation of Watermelon and Persimmon Fruit Residues to Obtain Compounds of Interest in Food Applications [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/191334 / Compendio

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No. of bitstreams: 1 cristianoferraraderesende.pdf: 2182147 bytes, checksum: a1f62e02cb7c97296c57ea6461357835 (MD5) Previous issue date: 2012-03-05 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Bromeliaceae é uma família essencialmente neotropical, cujos representantes são muito utilizados para fins paisagísticos. Uma das principais fontes de abastecimento do mercado consumidor de bromélias é o extrativismo o que, associado à destruição do ambiente natural, tem levado a perdas irreparáveis na biodiversidade da família, especialmente na Mata Atlântica, onde cerca de 40% das espécies encontram-se sob alguma categoria de ameaça. Pitcairnia encholirioides L. B. Sm. é uma espécie “criticamente em perigo” de extinção, sendo conhecida somente uma população dessa espécie, com cerca de 900 indivíduos, encontrada em 2004 em um afloramento rochoso muito degradado e sujeito ao fogo e ao pisoteio de animais, no município de Santa Maria Madalena, RJ. A utilização das técnicas de micropropagação pode reverter os riscos de extinção garantindo taxas elevadas de multiplicação, fornecendo material necessário ao mercado consumidor, evitando, dessa forma, o extrativismo das plantas nos seus locais de origem. A instalação e a conservação in vitro de bancos de germoplasma possuem especial importância, garantindo a sobrevivência de espécies raras e/ou endêmicas e fornecendo plantas para iniciativas de reintrodução. A etapa final da micropropagação é a aclimatização ex vitro, realizada após os procedimentos de multiplicação e enraizamento in vitro. A aclimatização é um período crítico para as plantas devido à perda de água, o que causa estresse hídrico e, como consequência, estresse oxidativo, podendo provocar problemas metabólicos e perdas elevadas. No presente trabalho, Pitcairnia encholirioides foi cultivada in vitro por 150 dias em meio de cultura adicionado de dois reguladores de crescimento (GA3 ou ANA), além de duas concentrações de sacarose (15 ou 30 g L-1) e sob dois tipos de vedação dos tubos de ensaio (vedação hermética com tampas e filme de PVC ou vedação com tampas que permitiam trocas gasosas), totalizando 8 tratamentos, período após o qual as plantas foram transferidas para condições ex vitro, em casa de vegetação, permanecendo por mais 180 dias. Dois desses tratamentos foram selecionados para as análises de desidratação, quando suas plantas foram submetidas a 30, 42 ou 54 dias sem irrigação, além do controle irrigado periodicamente, sendo posteriormente reidratadas durante 90 dias. Após os períodos de cultivo in vitro e permanência das plantas em casa de vegetação, foram realizadas análises dos conteúdos de prolina, proteínas totais, atividades das enzimas do metabolismo antioxidativo SOD, CAT, POD e PPO, além 2 dos teores de pigmentos fotossintetizantes. Além desses parâmetros, após desidratação e reidratação foram também avaliados os teores de carboidratos. De maneira geral, o cultivo in vitro em meio adicionado da menor concentração de sacarose e em tubos vedados hermeticamente se mostrou prejudicial, o que foi evidenciado pelo menor acúmulo de prolina, aumentos nas atividades das enzimas antioxidativas e menor acúmulo de pigmentos fotossintéticos. Após o período de aclimatização, não foram encontradas diferenças entre os tratamentos para a maioria dos parâmetros analisados, ou os dados oscilaram de maneira a não permitir uma generalização quanto aos efeitos residuais da sacarose, do tipo de tampa e dos reguladores de crescimento utilizados durante o cultivo in vitro. No período em que as plantas foram submetidas à desidratação progressiva, houve maior acúmulo de prolina e proteínas solúveis totais, além de aumento nos teores de pigmentos fotossintéticos nos tecidos provenientes de plantas cultivadas in vitro em meio contendo 30 g L-1 de sacarose e GA3. Houve também, em geral, queda mais acentuada nas atividades das enzimas em meio suplementado com a menor concentração de sacarose e ANA, além de oscilações nos teores de pigmentos. As relações entre os pigmentos sofreram leve redução em ambos os tratamentos, e os teores de carboidratos solúveis totais, sacarose, açúcares redutores e amido aumentaram com o prolongamento do estresse. Após a reidratação, todas as plantas demonstraram elevada capacidade de recuperação, apresentando em todas as análises realizadas valores muito similares aos das plantas do controle, não submetidas à desidratação. Os resultados obtidos no trabalho permitem concluir que o cultivo in vitro em meio adicionado de 30 g L-1 de sacarose e em tubos de ensaio que permitem trocas gasosas é o mais apropriado para plantas de P. encholirioides. Ademais, a concentração de sacarose adicionada ao meio de cultura também influencia o desenvolvimento das plantas após aclimatização, sendo encontrados melhores resultados em plantas cultivadas nos meios de cultura suplementados com a maior concentração desse carboidrato. Acredita-se que as plantas cultivadas nessas condições apresentariam maior capacidade de sobrevivência ao transplantio e maior resistência a períodos prolongados de estresse hídrico, aos quais, frequentemente, estão submetidas no ambiente natural. / Bromeliaceae is essentially a neotropical family, whose representatives are widely used for landscaping. One major source of supply for the consumer market is the extraction, which coupled with the destruction of the natural environment, has led to irreparable losses in biodiversity of the family, especially in the Atlantic Forest, where about 40% of bromeliad species are under some category of threat. Pitcairnia encholirioides L. B. Sm. is a critically endangered species. In 2004, a unique population of this species was found, with about 900 individuals in a very degraded rocky outcrop, subject to fire and trampling by animals, located in Santa Maria Madalena, RJ. The use of micropropagation can guarantee high multiplication rates, providing necessary material for the consumer market, effectively obviating the extraction of plants in their places of origin. In addition, installation and in vitro conservation of germplasm banks have special importance, ensuring the survival of rare species and/or endemic and providing micropropagated plants for reintroduction initiatives. Following the procedures of in vitro multiplication and rooting, the final stage of micropropagation is the ex vitro acclimatization, a critical period for the plants due to water loss, which causes water stress and, consequently, oxidative stress, which can cause metabolic problems and high losses. In this study, Pitcairnia encholirioides was grown for 150 days in vitro in a culture medium supplemented with two growth regulators (GA3 or NAA), and two concentrations of sucrose (15 or 30 g L-1) in two types of sealing of test tubes (hermetic seal with lids and PVC film and seal with lids that allowed gas exchange), totaling eight treatments, after which the plants were transferred to ex vitro conditions in a greenhouse, staying for more than 180 days. Two of these treatments were selected for analysis of dehydration when their plants were subjected to 30, 42 or 54 days without irrigation, apart from control irrigated periodically, being subsequently rehydrated for 90 days. After periods of in vitro cultivation and maintenance of the plants in the greenhouse, they were analyzed for proline content, total protein, activities of antioxidant metabolism enzymes SOD, CAT, POD and PPO, besides the content of photosynthetic pigments. Apart from these parameters, after dehydration and rehydration, levels of carbohydrates were also assessed. In general, the in vitro culture in medium containing the lowest concentration of sucrose and hermetically sealed tubes proved to be harmful, which was evidenced by lower levels of proline, increased 4 activity of antioxidant enzymes and lower accumulation of photosynthetic pigments. After the acclimatization period, no differences were found between treatments for most parameters, or data varied so as to not allow a generalization about the residual effects of sucrose, the type of cover and of the growth regulators used in the in vitro cultivation. During the period in which the plants were subjected to water stress, higher accumulation of proline and total soluble proteins occurred, and also increased levels of photosynthetic pigments in tissues from plants grown in vitro in medium containing 30 g L-1 sucrose and GA3. There was also, in general, sharper decrease in enzyme activity in medium with the lowest concentration of sucrose and NAA, as well as fluctuations in pigment. Relations between pigments suffered slight reduction in both treatments, and total soluble carbohydrates, sucrose, reducing sugars and starch increased with increasing stress. After rehydration, all plants showed high resilience, presenting for all analyzes values very similar to those of control plants not subjected to dehydration. The results indicate that the in vitro culture in medium supplemented with 30 g L-1 sucrose and tubes that allow gas exchange are the most appropriate. Moreover, the concentration of sucrose added to the culture medium also influences the development of plants after acclimatization, with best results found in plants grown in media supplemented with higher amount of this carbohydrate. It is believed that plants grown under these conditions would have higher capacity of resistance to transplanting and could survive for prolonged periods of water stress, which are often subjected in the natural environment.